ABSTRACT
Morus species had been used widely in the traditional medicines for various diseases. In this study we report the in vitro antiproliferative activity of the methanol extract of Morus alba. The extract is capable of inducing cytotoxicity in human colon cancer (HCT-15) cells (IC(50) = 13.8 µg/ml) and breast cancer (MCF-7) cells (IC(50) = 9.2 µg/ml), resulted in significant morphological changes of the cells, fragmentation of DNA, and caspase-3 activation- characteristics of apoptosis. It downregulated the amount of nitric oxide (NO) produced as a result of inducible nitric oxide (iNOS) activation. The HPLC analysis of the extract showed epicatechin (20%), myricetin (10%), quercetin hydrate (12%), luteolin (12%), and kaempferol (6%) as the major active components and ascorbic acid, gallic acid, pelargonidine, and p-coumaric acid as the minor components. To the best of our knowledge, this is the first report showing the downregulation of iNOS and induction of apoptosis by M. alba extract.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Breast Neoplasms/drug therapy , Colonic Neoplasms/drug therapy , Morus/chemistry , Nitric Oxide Synthase Type II/metabolism , Nitric Oxide/metabolism , Plant Extracts/pharmacology , Antioxidants/pharmacology , Apoptosis/drug effects , Breast Neoplasms/pathology , Caspase 3/metabolism , Cell Line, Tumor , Colonic Neoplasms/pathology , Female , Flavonoids/analysis , Humans , Inhibitory Concentration 50 , Kaempferols/analysis , Plant Extracts/analysis , Plant Extracts/chemistry , Plant Leaves/chemistryABSTRACT
Lectins represent a heterogeneous group of proteins/glycoproteins with unique carbohydrate specificity, with wide range of biomedical applications. The multi-step purification protocols generally used for purification of lectin result in a significant reduction in the final yield and activity. In the present study, Morus rubra lectin (MRL) was purified to homogeneity from the leaves using a single-step immobilized metal ion affinity chromatography (IMAC) procedure. The approximate molecular weight of purified MRL resolved as a single band on SDS-PAGE was 52 kDa. Final percentage yield of purified lectin by IMAC was calculated as 74.7 %. Purified MRL was specific to three sugars, galactose, D-galactosamine and N-acetyl-D-galactosamine, and rendered haemagglutination (HA) activity towards different human blood group RBCs. MRL showed stability over a wide range of temperature (up to 80 °C) and pH (4-11). Chelation of the lectin with EDTA did not alter HA which indicates that metal ion is not required for activity. In the presence of Fe(2+), Ca(2+), Zn(2+), Ni(2+), Mn(2+), Na(+) and K(+), HA activity was reduced to 50 %, whereas the presence of trivalent metal ions (Fe3(+) and Al(3+)) and Cu(2+) did not affect the activity. In the presence of Mg(2+) and Hg(2+), only 25 % of HA activity remained.
Subject(s)
Chemical Fractionation/methods , Chromatography, Affinity/methods , Metals/chemistry , Morus/chemistry , Plant Leaves/chemistry , Plant Lectins/isolation & purification , Carbohydrates/analysis , Hemagglutination/drug effects , Humans , Metals/pharmacology , Morus/cytology , Periodic Acid-Schiff Reaction , Plant Leaves/cytology , Plant Lectins/chemistry , Plant Lectins/pharmacologyABSTRACT
Medicinal values of mulberry are known to humans from ancient ages. The white mulberry, Morus alba L. is a rich source of many bioactive phytochemicals. Earlier investigations in our laboratory lead to the purification and characterization of an anti-proliferative lectin (MLL) from the leaves of this plant. Further to that, here we have investigated the mechanism of cell death induction by MLL on human breast cancer (MCF-7) and colon cancer (HCT-15) cells. Cells were treated with GI(50) concentration (concentration of lectin required for 50% inhibition of cell growth) of MLL (8.5 µg/ml for MCF-7 and 16 µg/ml for HCT-15) for 24 h to induce cell death. The induction of apoptosis was studied by morphological analysis, DNA fragmentation, apoptotic cell staining and caspase 3 activity assay. Apoptotic cells in sub G0-G1 phase were monitored using flow cytometry. MLL induced significant morphological changes and DNA fragmentation associated with apoptosis in MCF-7 and HCT-15 cells. Positive annexin V and acridine orange/ethidium bromide stained cells indicated apoptosis induction by MLL. Up-regulation of caspase 3 activity was also found in cells treated with MLL. Flow cytometry analysis showed an increase in the percentage of cells in sub G0-G1 phase confirming the MLL induced apoptosis. In conclusion, MLL induced apoptosis in MCF-7 and HCT-15 cells in a caspase dependent manner.
